Compositions and methods for early pregnancy diagnosis

ABSTRACT

Pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hoofed (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. By cloning expressed genes from ovine and bovine placental cDNA libraries, the inventors estimate that cattle, sheep, and most probably all ruminant Artiodactyla, possess possibly 100 or more PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Selected PAG that are products of the iOnvasive binucleate cells, expressed highly in early pregnancy at the time of trophoblast invasion and expressed weakly, if at all, in late gestation are useful in the early diagnosis of pregnancy. In a preferred embodiment, the invention relates to immunoassays for detecting these PAGs.

This application claims priority to U.S. Provisional Application SerialNo. 60/078,783 filed Mar. 20, 1998 and U.S. Provisional ApplicationSerial No. 60/106,188 filed Oct. 28, 1998. The entire text of each ofthe above-referenced disclosures is specifically incorporated byreference herein without disclaimer. The government may own rights inthe present invention pursuant to grant R37 HD29483 and USDA grant9601842.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of veterinarymedicine, reproductive biology and diagnostics. More specifically, thepresent invention relates to the use of analytical methods to detectearly stage pregnancy.

II. Related Art

Pregnancy diagnosis is an important component in sound reproductivemanagement, particularly in the dairy industry (Oltenacu et al., 1990)where a high proportion of artificial inseminatioris fail (Streenan andDiskin, 1986). A reliable yet simple pregnancy test for cattle has longbeen sought. Several procedures are available, including a milkprogesterone assay (Oltenacu et al., 1990; Markusfeld et al., 1990)estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al.,1995), rectal palpation (Hatzidakis et al., 1993), ultrasound (Beal etal., 1992; Cameron and Malmo, 1993), and blood tests forpregnancy-specific antigens. Of these, the progesterone milk assay isthe most cost effective for the producer (Oltenacu et al., 1990;Markusfeld et al., 1990). Next best is rectal palpation, performed atday 50 (Oltenacu et al., 1990). Even though all the procedures arepotentially useful, all have fallen short of expectations in terms oftheir practical, on-farm use. For example, measurements of milk or serumprogesterone around day 18-22 yield unacceptably high rates of falsepositives (Oltenacu et al., 1990; Markusfeld et al., 1990). Rectalpalpation can be used to detect pregnancy as early as day 35, but thisprocedure can lead to 5-10% or greater embryonic mortality (Oltenacu etal., 1990; Hatzidakis et al., 1993). Rectal palpation on day 50 causesless damage to the embryos, but had only marginal economic value becauseof its lateness (Oltenacu et al., 1990). Ultrasonography has anadvantage over rectal palpation in accuracy, particularly before day 45(Beal et al., 1992; Cameron and Malmo, 1993), but the instrument isexpensive, its use requires considerable training, and there is a finiterisk to the animal. A related procedure, Doppler sonography, is moreaccurate than rectal palpation (Cameron and Malmo, 1993), but againrequires well trained personnel. The presence of estrone sulfate inurine or serum provides another test but is only useful after day 100 asconcentrations rise (Holdsworth et al., 1982; Warnick et al., 1995).

The discovery of pregnancy-specific protein B (PSP-B) (Butler et al.,1982) provided a new approach to pregnancy diagnosis since it could bedetected in the blood of pregnant cows by the fourth week of pregnancy(Sasser et al., 1986; Humblot et al., 1988). Two other groups havedeveloped immunoassays that may be based on an identical orimmunologically similar antigen (Zoli et al., 1992a; Mialon et al.,1993; Mialon et al., 1994). In one case, the antigen (Mr ˜67 kDa) wascalled bovine pregnancy-associated glycoprotein (boPAG; now boPAG-1)(Zoli et al., 1992a); in the second, it was designated as pregnancyserum protein 60 (PSP60) (Mialon et al., 1993; Mialon et al., 1994). Theimmunoassay for PSP-B/boPAG1/PSP60 has two advantages. First, it candetect pregnancy relatively early. Second, interpretation of the assaysdoes not require knowledge of the exact date of service, since boPAG-1immunoreactive molecules are always present in the maternal serum ofpregnant cows by day 28, and concentrations increase as pregnancyadvances (Sasser et al., 1986; Mialon et al., 1993; Mialon et al.,1994).

There remain, however, two major disadvantages to this procedure. First,positive diagnosis in the fourth week of pregnancy remains somewhatuncertain because antigen concentrations in blood are low and somewhatvariable. Second, boPAG1 concentrations rise markedly at term (Sasser etal., 1986; Zoli et al., 1992a; Mialon et al., 1993) and, due to the longcirculating half-life of the molecule (Kiracofe et al., 1993), theantigen can still be detected 80-100 day postpartum (Zoli et al., 1992a;Mialon et al., 1993; Mialon et al., 1994; Kiracofe et al., 1993),compromising pregnancy diagnosis in cows bred within the earlypostpartum period. Thus, the test can be carried out in dairy cows atday 30 only if artificial insemination (“AI”) is performed at or after70 day post-partum.

A pregnancy test that could be carried out reliably and early inpregnancy could provide definitive indication as to whether rebreedingor culling is required. In general, AI is successful less than 50% ofthe time and the producer must either rely on overt signs of return toestrus (that are easily missed) or delay rebreeding until pregnancyfailure is confirmed by one of the methods described above. Such delaysare extremely costly and constitute a major economic loss to theindustry. In the North Island of New Zealand alone, over two millioncows are bred in a six-week period. A precise knowledge of the pregnancystatus of these animals would be an invaluable aid to that and otherdiary industries worldwide. As should be apparent, this field has a needfor a feasible, sensitive and accurate pregnancy test in cattle that canbe performed by the end of the third week after insemination.

SUMMARY OF THE INVENTION

Therefore, it is an objective of the present invention to provide asensitive and accurate test for early pregnancy. Using a selected boPAGas the biochemical marker, the present invention provides an earlypregnancy test in which the PAG antigen a) is produced abundantly inearly, and preferably not in late, pregnancy, b) is a product of thebinucleate cell, and absent or not present in significant amountspostpartum, and c) minimally cross-reacts with late PAG products thatmight persist in maternal serum during the post-partum interval. Theearly immunoassay will be particularly useful in the dairy industrywhere animals are usually confined for at least part of the day andwhere intensive management is practiced. A modified test also is likelyto have value in captive breeding programs for other animals, e.g., forthe ruminants okapi or giraffe and possibly for other non-ruminantspecies.

Thus, in a particularly preferred embodiment, there is provided a methodfor detecting pregnancy in a bovine animal comprising obtaining a samplefrom the animal; and detecting at least one of pregnancy associatedantigen (PAG) wherein the PAG is present in early pregnancy and absentat about two months postpartum, whereby the presence of the PAGindicates that the animal is pregnant. Insemination is usually, but notinvariably, performed about two months after calving in dairy cattle,until a successful conception results. The detection method may beapplied within about 15 days of insemination and advantageously at about20 to about 25 days after insemination. Given these facts, the timewindow for the disappearance of a useful PAG is about two months aftercalving, although earlier disappearance is also advantageous. However,PAGs which persist until about 65, about 70, about 75, about 80, about85, about 90, about 95, or about 100 days after calving also aresuitable for use. The exact day for this determination may varydepending on individual circumstances, however, given the teachingsprovided herein, an individual of skill in the art will understand thesignificance of testing for the absence of PAG during this time periodand will be able to determine such a day. For example, if inseminationoccurs at a later date than 60 days post-partum, PAGs with a laterdisappearance profile may be useful. Thus, it is contemplated that thePAG of the present invention is detectable in early pregnancy but is notdetectable at two months postpartum. Also, it is understood that the PAGindicative of early pregnancy may be absent in late pregnancy or presentin amounts that are markedly less than those found in early pregnancy(for example, between day 15 and day 30 of pregnancy).

In particularly preferred embodiments, the PAG may be selected from thegroup consisting of PAG2, PAG4, PAG5, PAG6, PAG7 and PAG9. In morepreferred embodiments, the PAG, independently, may be BoPAG2, BoPAG4,BoPAG5, BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16;boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21.

In particular aspects of the present invention, the sample may be asaliva, serum, blood, milk or urine sample. Methods of sample collectionare well known to those of skill in the art, for example, blood may becollected by needle from a tail vein or other blood vessel, milkwithdrawn from the udder. Saliva and urine also may be collectedaccording to well known techniques. In defined embodiments, it iscontemplated that the detecting comprises an immunologic detection. Inpreferred embodiments, the immunologic detection comprises detectionBoPAG2, BoPAG4, BoPAG5, BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v;boPAG15; boPAG16; boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21 withpolyclonal antisera. In alternative embodiments, the immunologicdetection comprises detection of BoPAG2, BoPAG4, BoPAG5, BoPAG6, BoPAG7,BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16; boPAG17; boPAG18; boPAG19;boPAG20 or boPAG21 with a monoclonal antibody in particularly preferredembodiment, the immunologic detection may comprise ELISA, in otherembodiments, the immunologic detection may comprises RIA, in stillfurther alternative embodiments, the immunologic detection comprisesWestern blot.

In certain aspects of the present invention, the method for detectingpregnancy may further comprise detecting a second PAG in the sample. Thesecond PAG may be selected from the group consisting of BoPAG2, BoPAG4,BoPAG5, BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16;boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21. Alternatively, the secondPAG may be any other pregnancy Likewise the present inventioncontemplates a pregnancy detection method that further comprisesdetecting a third PAG in the sample.

In those embodiments employing ELISA as an immunological technique, itis contemplated that the ELISA may be a sandwich ELISA comprisingbinding of a PAG to a first antibody preparation fixed to a substrateand a second antibody preparation labeled with an enzyme. Sandwich ELISAis well known to those of skill in the art. In particularly preferredembodiments, the enzyme may be alkaline phosphatase or horseradishperoxidase. In other embodiments, the first antibody preparation may bea monoclonal antibody preparation.

Other aspects of the present invention contemplate an antibodycomposition that reacts immunologically with BoPAG2, BoPAG4, BoPAG5,BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16; boPAG17;boPAG18; boPAG19; boPAG20 or boPAG21. Particularly preferred embodimentscontemplate an provide an antibody composition that reactsimmunologically with BoPAG4. Further embodiments provide an antibodycomposition that reacts immunologically with BoPAG5. Still furtherembodiments contemplate an antibody composition that reactsimmunologically with BoPAG6. Other embodiments contemplate an antibodycomposition that reacts immunologically with BoPAG7. Still furtherembodiments, contemplate an antibody composition that reactsimmunologically with BoPAG9. It is contemplated that the antibodycomposition. may be a monoclonal antibody composition or a polyclonalantibody composition.

The present invention further provides a hybridoma cell that secretes amonoclonal antibody that reacts immunologically with BoPAG2, BoPAG4,BoPAG5, BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16;boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21.

Also contemplated herein is a method of making a monoclonal antibody toBoPAG2, BoPAG4, BoPAG5, BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v;boPAG15; boPAG16; boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21comprising the steps of immunizing an animal with a BoPAG preparation;obtaining antibody secreting cells from the immunized animal;immortalizing the antibody secreting cells; and identifying animmortalized cell that secretes antibodies that bind immunologicallywith the immunizing BoPAG.

Another aspect of the present invention provides a method of identifyinga pregnancy associated glycoprotein (PAG) that is an early indicator ofpregnancy in an Eutherian animal comprising the steps of obtaining acDNA library prepared from the placenta of the animal between days 15and 30 of pregnancy; and hybridizing the library under high stringencyconditions with a PAG-derived nucleic acid probe; whereby hybridizationof the probe identifies the PAG.

Also provided by the present invention is a method of identifying apregnancy associated glycoprotein (PAG) that is an early indicator ofpregnancy in an Eutherian animal comprising the steps of obtaining anRNA preparation from the placenta of the animal between days 15 and 30of pregnancy; and performing RT-PCR™ on the preparation usingPAG-derived primers; whereby amplification identifies the PAG.

In particularly preferred embodiments, the PAG detected in cattle (Bostaurus) may be any one or more of the following PAGs that are so farknown to be produced in early pregnancy, namely: BoPAG2, BoPAG4, BoPAG5,BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16; boPAG17;boPAG18; boPAG19; boPAG20 or boPAG21. More specifically, the bovine PAGsthat may be detected comprise the sequence of one or more of thefollowing amino acid sequences: SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32; SEQ ID NO:40; SEQID NO:42; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:48; SEQ ID NO:50; SEQ IDNO:52; SEQ ID NO:54; SEQ ID NO:56. When applied to other species, thepresent invention will allow detection of other PAGs produced at thetime the trophoblast (pre-placenta) begins either to attach or toimplant into the uterine wall of the mother. The “early” PAGs in thesespecies may cross-react immunologically with the PAGs useful indetecting early pregnancy in cattle.

The present invention contemplates an isolated and purified BoPAG2polypeptide. In preferred embodiment, the BoPAG2 polypeptide comprisesthe sequence of SEQ ID NO:25. Further, the invention contemplates anisolated and purified BoPAG4 polypeptide. In particularly preferredembodiments, the BoPAG4 polypeptide comprises the sequence of SEQ IDNO:27. Another embodiment contemplates an isolated and purified BoPAG5polypeptide. A particularly preferred BoPAG5 polypeptide comprises thesequence of SEQ ID NO:28. Yet another embodiment provides an isolatedand purified BoPAG6 polypeptide. In preferred embodiments, the BoPAG6polypeptide comprises the sequence of SEQ ID NO:29. Another embodimentcontemplates an isolated and purified BoPAG7 polypeptide. An especiallypreferred BoPAG7 polypeptide comprises the sequence of SEQ ID NO:30.Further contemplated by the present invention is an isolated andpurified BoPAG9 polypeptide. In preferred embodiments, the BoPAG9polypeptide comprises the sequence of SEQ ID NO:32. Further contemplatedby the present invention is an isolated and purified BoPAG7vpolypeptide. In preferred embodiments, the BoPAG7v polypeptide comprisesthe sequence of SEQ ID NO:40. Further contemplated by the presentinvention is an isolated and purified BoPAG9v polypeptide. In preferredembodiments, the BoPAG9v polypeptide comprises the sequence of SEQ IDNO:42. Further contemplated by the present invention is an isolated andpurified BoPAG15 polypeptide. In preferred embodiments, the BoPAG15polypeptide comprises the sequence of SEQ ID NO:44. Further contemplatedby the present invention is an isolated and purified BoPAG16polypeptide. In preferred embodiments, the BoPAG16 polypeptide comprisesthe sequence of SEQ ID NO:46. Further contemplated by the presentinvention is an isolated polypeptide. In preferred embodiments, theBoPAG16 polypeptide comprises the and purified BoPAG17 polypeptide. Inpreferred embodiments, the BoPAG17 polypeptide comprises the sequence ofSEQ ID NO:48. Further contemplated by the present invention is anisolated and purified BoPAG18 polypeptide. In preferred embodiments, theBoPAG18 polypeptide comprises the sequence of SEQ ID NO:50. Furthercontemplated by the present invention is an isolated and purifiedBoPAG19 polypeptide. In preferred embodiments, the BoPAG19 polypeptidecomprises the sequence of SEQ ID NO:52. Further contemplated by thepresent invention is an isolated and purified BoPAG20 polypeptide. Inpreferred embodiments, the BoPAG20 polypeptide comprises the sequence ofSEQ ID NO:54. Further contemplated by the present invention is anisolated and purified BoPAG2 1 polypeptide. In preferred embodiments,the BoPAG21 polypeptide comprises the sequence of SEQ ID NO:56.

Alternative embodiments of the present invention define an isolated andpurified nucleic acid encoding BoPAG2. In particularly preferredembodiments, the BoPAG2 encoding nucleic acid comprises the sequence ofSEQ ID NO:2. In other preferred embodiments, the BoPAG2 encoding nucleicacid encodes a BoPAG2 polypeptide comprising the sequence of SEQ IDNO:25.

Another embodiment provides an isolated and purified nucleic acidencoding BoPAG4. In preferred embodiments the BoPAG4 encoding nucleicacid comprises the sequence of SEQ ID NO:4. In other equally preferredembodiments, the BoPAG4 encoding nucleic acid encodes a BoPAG4polypeptide comprising the sequence of SEQ ID NO:27.

In yet another embodiment, there is contemplated an isolated andpurified nucleic acid encoding BoPAG5. In preferred embodiments, theBoPAG5 encoding nucleic acid comprises the sequence of SEQ ID NO:5. Inother preferred embodiments, the BoPAG5 encoding nucleic acid encodes aBoPAG5 polypeptide comprising the sequence of SEQ ID NO:28.

In still another aspect of the present invention there is provided anisolated and purified nucleic acid encoding BoPAG6. In particularlypreferred aspects the BoPAG6 encoding nucleic acid comprises thesequence of SEQ ID NO:6. In particularly preferred embodiments, thenucleic acid encodes a BoPAG6 polypeptide comprising the sequence of SEQID NO:29.

Also contemplated by the present invention is an isolated and purifiednucleic acid encoding BoPAG7. In preferred embodiments, the nucleic acidcomprises the sequence of SEQ ID NO:7. In other preferred embodiments,the nucleic acid encodes a BoPAG7 polypeptide comprising the sequence ofSEQ ID NO:30.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG9. In particular embodiments the BoPAG9 encodingnucleic acid comprises the sequence of SEQ ID NO:9. In otherparticularly preferred embodiments, the BoPAG9 encoding nucleic acidencodes a BoPAG9 polypeptide comprising the sequence of SEQ ID NO:32.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG7v. In particular embodiments the BoPAG7v encodingnucleic acid comprises the sequence of SEQ ID NO:39. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG7v polypeptide comprising the sequence of SEQ ID NO:40.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG9v. In particular embodiments the BoPAG9v encodingnucleic acid comprises the sequence of SEQ ID NO:41. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG9v polypeptide comprising the sequence of SEQ ID NO:42.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG15. In particular embodiments the BoPAG15 encodingnucleic acid comprises the sequence of SEQ ID NO:43. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG15 polypeptide comprising the sequence of SEQ ID NO:44.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG16. In particular embodiments the BoPAG16 encodingnucleic acid comprises the sequence of SEQ ID NO:45. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG16 polypeptide comprising the sequence of SEQ ID NO:46.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG17. In particular embodiments the BoPAG17 encodingnucleic acid comprises the sequence of SEQ ID NO:47. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG17 polypeptide comprising the sequence of SEQ ID NO:48.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG18. In particular embodiments the BoPAG18 encodingnucleic acid comprises the sequence of SEQ ID NO:49. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG18 polypeptide comprising the sequence of SEQ ID NO:50.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG19. In particular embodiments the BoPAG19 encodingnucleic acid comprises the sequence of SEQ ID NO:51. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG19 polypeptide comprising the sequence of SEQ ID NO:52.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG20. In particular embodiments the BoPAG20 encodingnucleic acid comprises the sequence of SEQ ID NO:53. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG20 polypeptide comprising the sequence of SEQ ID NO:54.

Yet another embodiment contemplates an isolated and purified nucleicacid encoding BoPAG21. In particular embodiments the BoPAG21 encodingnucleic acid comprises the sequence of SEQ ID NO:55. In otherparticularly preferred embodiments, the BoPAG7v encoding nucleic acidencodes a BoPAG21 polypeptide comprising the sequence of SEQ ID NO:56.

Also contemplated herein are oligonucleotides comprising at least 15consecutive base pairs of any PAG encoding sequence, or a complementthereof, disclosed herein. Particularly contemplated is anoligonucleotide comprising at least about 15 consecutive bases of thesequence of SEQ ID NO:9, or the complement thereof. In otherembodiments, the oligonucleotide is about 20 bases in length. Alsocontemplated is an oligonucleotide comprising at least about 15consecutive bases of the sequence of SEQ ID NO:7, or the complementthereof. another embodiments contemplates an oligonucleotide comprisingat least about 15 consecutive bases of the sequence of SEQ ID NO:6, orthe complement thereof. Yet another embodiments provides anoligonucleotide comprising at least about 15 consecutive bases of thesequence of SEQ ID NO:5, or the complement thereof. In still a furtherembodiment, there is contemplated an oligonucleotide comprising at leastabout 15 consecutive bases of the sequence of SEQ ID NO:4, or thecomplement thereof. Yet another embodiment contemplates anoligonucleotide comprising at least about 15 consecutive bases of thesequence of SEQ ID NO:2 or the complement thereof Yet another embodimentcontemplates an oligonucleotide comprising at least about 15 consecutivebases of the sequence of SEQ ID NO:39 or the complement thereof. Yetanother embodiment contemplates an oligonucleotide comprising at leastabout 15 consecutive bases of the sequence of SEQ ID NO:41 or thecomplement thereof Yet another embodiment contemplates anoligonucleotide comprising at least about 15 consecutive bases of thesequence of SEQ ID NO:43 or the complement thereof. Yet anotherembodiment contemplates an oligonucleotide comprising at least about 15consecutive bases of the sequence of SEQ ID NO:45 or the complementthereof. Yet another embodiment contemplates an oligonucleotidecomprising at least about 15 consecutive bases of the sequence of SEQ IDNO:47 or the complement thereof. Yet another embodiment contemplates anoligonucleotide comprising at least about 15 consecutive bases of thesequence of SEQ ID NO:49 or the complement thereof. Yet anotherembodiment contemplates an oligonucleotide comprising at least about 15consecutive bases of the sequence of SEQ ID NO:51 or the complementthereof. Yet another embodiment contemplates an oligonucleotidecomprising at least about 15 consecutive bases of the sequence of SEQ IDNO:53 or the complement thereof. Yet another embodiment contemplates anoligonucleotide comprising at least about 15 consecutive bases of thesequence of SEQ ID NO:55 or the complement thereof. Of course it isunderstood that oligonucleotides of longer lengths are also contemplatedincluding oligonucleotides of 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50 or more consecutive base pairs in length.

The present invention further provides a kit comprising a firstmonoclonal antibody preparation that binds immunologically to BoPAG2,BoPAG4, BoPAG5, BoPAG6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15;boPAG16; boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21; and a suitablecontainer means therefor. It is contemplated that in particularembodiments, the kit may further comprise a second monoclonal antibodypreparation that binds immunologically to the same BoPAG as the firstmonoclonal antibody, but wherein the first and the second monoclonalantibodies bind to different epitopes; and a suitable container meanstherefor. In particularly preferred aspects the first antibodypreparation is attached to a support. It is contemplated that thesupport may be any support routinely used in immunological techniques.In particularly preferred embodiments, the support independently is apolystyrene plate, test tube or dipstick.

In particular embodiments, the second antibody preparation comprises adetectable label. The detectable label may be independently afluorescent tag, a chemilluminescent tag, or an enzyme. In particularlydefined embodiment, the enzyme is alkaline phosphatase or horseradishperoxidase. In further preferred embodiments, the kit may also comprisea substrate for the enzyme. In other embodiments, the kit may furthercomprise a buffer or diluent; and a suitable container means therefor.

In another embodiment, there is provided a kit including a firstantibody composition that binds immunologically to BoPAG2, BoPAG4,BoPAG5, BoPA6, BoPAG7, BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16;boPAG17; boPAG18; boPAG19; boPAG20 or boPAG21; and a suitable containermeans therefor as well as a second antibody composition that bindsimmunologically to the same boPAG as the first antibody composition, butthe first and second antibody compositions bind to different epitopes;and included in this defined kit is a suitable container means therefor.More specifically, this aspect of the invention encompasses a secondantibody composition including a detectable label. Other kit components,including reagent reservoirs, instructions and the like are well knownto those of skill in the art and also are contemplated for use in thekits described herein.

In other embodiments, there is provided a method for detecting pregnancyin a non-bovine Eutherian animal comprising obtaining a sample from theanimal; and detecting at least one of pregnancy associated antigen (PAG)in the sample, wherein the PAG is present in early pregnancy, wherebythe presence of the PAG indicates that the animal is pregnant. The PAGmay be absent at a period postpartum. As used herein, the term “absent”means not present using a given detection method. In other embodimentsthe PAG may be diminished postpartum. As used herein, “diminished” meansdropping to undetectable or almost undetectable levels using a givenprotocol. In particularly preferred embodiment, the PAG may be selectedfrom the group consisting of PAG2, PAG4, PAG5, PAG6, PAG7 and PAG9. Invarious embodiments, the animal in which pregnancy is being determined,may include all Artiodactyla which include Suidae (pigs and theirrelatives) and Camellidae (camels). It is contemplated that the animalmay be a member of the suborder Ruminantia. In more defined embodiments,the Ruminantin may be a member of the family Bovidae. In more particularembodiments, the animal is a goat or sheep. In other embodiments theanimal may be a member of the order Perissodactyla. In preferredembodiments, the animal may be a horse or rhinoceros. In alternativepreferred embodiments, the animal is a member of the order Carnivora.More particularly the animal may be an animal of the canine or felinespecies. Even more particularly, the animal may be a dog or a cat. Inother embodiments, the animal may be a human or a panda.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Aligned amino acid sequences of different boPAGs. Each structurewas inferred from the sequences of its cDNA. The likely signal sequenceis underlined and a known site of propeptide sequence cleavage(ISG↓RG/DS) for certain PAGs is shown (vertical arrow). Many additionalsequences, some from cDNA not containing entire ORF, others differingless than 5% in nucleotide sequence from those shown, are known.Numbering at end of rows is by amino acid residue starting the Metl.Numbers in parentheses show the equivalent residue of pepsin. Boxesindicate the conserved sequences around the catalytic aspartic acidresidues (Asp32 and Asp 215). GenBank Accession codes for boPAG1 throughboPAG12 are M73961, L06 151, L06 153 and AF020506 through AF 020514,respectively.

FIG. 2. The aligned amino acid sequences of different ovPAGs. See legendto FIG. 1 for details. GenBank Accession codes for ovPAG1 through ovPAG9are M73962, U30251 and U94789 through U94795, respectively.

FIG. 3. Summary of cloning data for boPAG expressed in day 19 and 25bovine placenta. Early boPAG clones were identified by three independentprocedures. Numbers indicate how many clones of identical sequence wereisolated by each procedure. First, a day 25 bovine cDNA library wasscreened by homologous hybridization (Hybrid) with a probe consisting ofov, bo and poPAG1 and 2 as well as eqPAG cDNA. Sixteen clones with fulllength cDNA were purified and partially sequenced. The library was thenimmunoscreened (Immuno) with and anti-boPAG1antiserum and 19 clones werepurified and partially sequenced. RNA from a day 19 Holstein cowplacenta was reverse transcribed and amplified with PCR™ (RT-PCR™). ThePCR™ products were subcloned and partially sequenced. Note, most of theearly boPAG were identified by homologous hybridization.

FIG. 4. Pairwise companions of the amino acid and nucleotide sequencesof bovine PAG The data show percent nucleotide sequence identity(shaded) and percent amino acid sequence identity of translatedsequences (unshaded).

FIG. 5. A phylogram based on amino acid sequences showing therelationship of all known cloned PAGs to common mammalian asparticproteinases. The tree was constructed by the Wisconsin GCG programsDistances and GrowTree. The lengths of the branches are proportional tothe degree of amino acid diversity within pairs of proteins. Proteindata bank symbols: PEPA_pig, porcine pepsinogen A; PEPF_rabbit, rabbitpepsinogen F.

FIG. 6. Southern genomic blotting of DNA from some selected ruminant andnonruminant ungulate species and from a member of the family Carnivora(Panda). DNA was digested with EcoRI and probed with a boPAG1 probe. DNAsize markers are on the left. Some samples of DNA, e.g., Suffolk Sheepand Mule Deer were analyzed twice.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The PresentInvention

Despite the availability of several assays to detect pregnancy, thereremains a need to provide improved assays for accurate and earlydetection of pregnancy, especially in cattle that are bred within two tothree months postpartum or earlier. In the context of the presentinvention, a preferred species is bovine. The present inventionidentifies several placentally expressed polypeptides, designatedpregnancy associated glycoproteins (PAGs) that can be utilized to makeearly and accurate diagnoses of bovine and other pregnancies. Additionalembodiments include the development of reagents from these polypeptides,and their corresponding genes, for use in assays to detect pregnancy.Extrapolation to other closely and distantly related species extends theapplication of these methods.

For use according to the present invention, selected PAGs are those thata) is produced abundantly in early and preferably not in late pregnancy,b) is a product of the binucleate cell, and absent or not present insignificant amounts postpartum, and c) minimally cross-reacts with latePAG products that might persist in maternal serum during the post-partuminterval. Further, the PAG should be detectable in serum atconcentrations sufficient for a straightforward and rapid detection.Finally, the PAGs should be amenable to reproducible polyclonal andmonoclonal antibody production in suitable host species. The remainingdisclosure describes various features of the invention and theirimplementation.

II. Pregnancy Associated Glycoproteins

The placenta is the hallmark of the eutherian mammal. Rather than beingthe most anatomically conserved mammalian organ, however, it arguably isthe most diverse (Haig, 1993). Placentation ranges from the invasivehemochorial type, as in the human, where the trophoblast surface is indirect contact with maternal blood, to the epitheliochorial (e.g., pig),where the uterine epithelium is not eroded (Amoroso, 1952). Not only isplacental structure highly variable, the polypeptide hormones theplacenta produces also vary between species (Haig, 1993; Roberts et al.,1996). For example, no group of mammals other than higher primatespossesses a chorionic gonadotrophin homologous to hCG for luteal supportin early pregnancy, and only the ruminant ungulates are known to produceType I interferon as an antilyteolytic hormone (Roberts et al., 1996).

Placentation in ruminants, such as cattle and sheep, is superficial,relatively noninvasive, and known as synepitheliochorial cotyledonary(Wooding, 1992). ‘Synepitheliochorial’ describes the fetal-maternalsyncytium formed by the fusion of trophoblast binucleate cells anduterine epithelial cells, whereas, ‘cotyledonary’ describes the grossstructure of the placenta and specifically the tufts of villoustrophoblast (cotyledons) that insinuate themselves into the crypts ofthe maternal caruncles. These regions of interdigitated and partiallyfused fetal cotyledonary and maternal caruncles are the placentomes andare the main sites for nutrient and gas exchange in the placenta. Thebinucleate cells, which compose about 20% of the surface epithelium(trophectoderm) migrate and fuse with maternal uterine epithelial cellsand deliver their secretory products directly to the maternal system.Among the products are the placental lactogens (Wooding, 1981) and thepregnancy-associated glycoproteins (Zoli et al., 1992a.)

Bovine pregnancy-associated glycoproteins (boPAGs), also known under avariety of other names including pregnancy-specific protein-B (Butler etal., 1982), were discovered in attempts to develop pregnancy tests forlivestock (Sasser et al., 1986; Zoli et al., 1991; Zoli et al., 1992a).Rabbits were injected with extracts of placental cotyledons, andantibodies not directed against placental antigens were removed byadsorption with tissue extracts from nonpregnant animals. The resultingantisera provided the basis of an accurate pregnancy test for cattle andsheep as early as one month post-insemination.

Xie et al. (1991) used an antiserum directed against purified boPAGsfrom cattle and from sheep to screen cDNA libraries from late placentaltissue. The full-length cDNAs shared 86% nucleotide sequence identitieswith each other and a surprising 60% sequence identity to pepsinogens.The boPAGs had mutations in and around their active sites that wouldrender them inactive as proteinases (Xie et al., 1991; Guruprasad etal., 1996). The similarities to pepsin A (˜50% amino acid identity) andchymosin (˜45%) in primary structure has allowed atomic models of ovine(ov)PAG1 and boPAG1 to be built (Guruprasad et al., 1996). Bothmolecules have the bibbed structure typical of all known eukaryoticaspartic proteinases and possess a cleft between the two lobes capableof accommodating peptides up to 7 amino acids long. Modeling stronglysuggested that both ovPAG1 and boPAG1 can bind the pepsin inhibitorpepstatin, a prediction that has been validated.

Even in initial studies (Butler et al., 1982; Zoli et al., 1991; Xie etal., 1991; Xie et al., 1994; Xie et al., 1996), it was clear that theboPAGs were heterogenous in molecular weight and charge, and as moreisoforms have been purified it has become evident that they differ intheir amino terminal sequences (Atkinson et al., 1993; Xie et al.,1997a). Further library screening has revealed additional transcripts inruminants (Xie et al., 1994; Xie et al., 1995; Xie et al., 1997b) andthe existence of PAGs in non-ruminant species such as the pig(Szafranska et al., 1995), and the horse (Guruprasad et al., 1996).

Despite their apparent lack of proteolytic activity, all of the PAGswhose amino terminal sequences have been determined are proteolyticallyprocessed in a manner typical of other aspartic proteases such as pepsin(Davies, 1990). For example, a pro-peptide of most PAGs, whichconstitutes the first 38 amino acids of the secreted form and whichnormally folds into the active site region, has been cleaved from thesecreted forms of PAG. Thus, the calculated molecular weight of themature, non-glycosylated PAG, i.e. with signal sequence propeptideremoved would be ˜36,000 daltons and the circulating antigen in serumwould also lack this segment. The observed molecular weight of secretedPAG, however, is much larger ranging from 45,000 daltons to 90,000daltons (Xie et al., 1991; Sasser et al., 1989; Xie et al., 1996),probably due to extensive glycosylation (Holdsworth et al., 1982).Multiple boPAG genes in the bovine genome have most likely contributedto the triphasic alterations of PAG concentrations in maternal serum.

A. BoPAG1

Bovine (bo) PAG1 was initially identified as a unique placental antigenby raising antisera to total bovine placental extracts (Zoli et al.,1991). It is a product of binucleate trophoblast cells (Xie et al.,1991; Zoli et al., 1992b) which constitute the invasive component of theplacenta (Wooding, 1992; Guillomot, 1995). In 1991, cDNA for both boPAG1and ovine PAG1 was identified (ovPAG1) (Xie et al., 1991). Surprisingly,the PAG1 belong to the aspartic proteinase (AP) gene family, a groupingthat includes pepsin, chymosin, renin, and cathepsin D and E (Guruprasadet al., 1996). Unlike other members of the AP family, both ovPAG1 andboPAG1 appear to be enzymatically inactive, since the catalytic domainin the active site region is mutated (Xie et al., 1991; Guruprasad etal., 1996).

BoPAG1 gene contains 9 exons and 8 introns (Xie et al., 1996), anidentical organization to that of other mammalian aspartic genes.Southern genomic blotting with a probe encompassing exon 7 and exon 8,which represent the most conserved region of PAG relative to other AP,indicated that there were probably many PAG genes. In addition, when abovine genomic library was probed with boPAG1 cDNA, 0.06% positive phageplaques were identified, suggesting that there may be 100 or more PAGgenes in the bovine genome (Xie et al., 1995). This approximation hasrecently been confirmed by a variety of other approaches (Xie et al.,1997b).

Levels of boPAG1 or related molecules that cross-react with a boPAG-1antiserum are very low around day 21 to day 27 (Warnick et al., 1995;Beal et al., 1992; Cameron and Malmo, 1993; Butler et al., 1982), aremaintained at a higher, but still low concentration until about day 100of the pregnancy and then rise quickly to ˜100 ng/ml. The concentrationsthen remain relatively constant until the last quarter of pregnancy whenthey peak at 1 μg/ml of serum or greater right before parturition. Oneexplanation for the triphasic profile of boPAG1 immunoreactivity is thatexpression of boPAG1 is very low in early pregnancy, rises considerablyat mid gestation and peaks before parturition (Sasser et al., 1986; Zoliet al., 1992a; Patel et al., 1995). Alternatively, the presence ofimmunoreactive antigen in very early pregnancy may be due to theproduction of other boPAGs. The rise in the second trimester may reflectproduction of yet a different class of boPAG or possibly the initiationof low PAG1 expression. The exponential rise of boPAGs just prior toparturition could represent a sudden increase in the synthesis of one ormore boPAG1 related molecules or increased “escape” across a leakierutero-placental junction.

Immunocytochemistry and in situ hybridization analyses have shown thatboPAG1 and ovPAG1, and their close relatives (since neither the antiseranor the probes are expected to be monospecific) are localized tobinucleate cells (Xie et al., 1991; Zoli et al., 1992b) In contrast, theantigenically distinct boPAG2 is expressed in predominantly mononucleatecells of the trophectoderm (Xie et al., 1994). In the ruminants,binucleate cells are the invasive components of the trophoblast and donot appear until about day 13 in sheep and day 17 in cattle (Wooding,1992). They then quickly increase in number. By day 21 in cattle theyconstitute up to 20% of cells in the trophectoderm, and a highpercentage are actively fusing with maternal uterine epithelial cells(Wooding, 1992; King et al., 1980; Guillomot, 1995). Binucleate cellgranules, which contain PAG1 (Zoli et al., 1992b), are discharged fromthe fusion cell towards the maternal stroma and its network ofcapillaries. Therefore, the binucleate cell products have ready accessto the maternal circulation.

B. Novel OvPAG and BoPAG Species

According to the present invention, cDNA for a series of novel boPAGshave been identified and cloned (FIG. 1). A similar large family ofovine (ov) PAGs have been identified from sheep placenta (Xie et al.,1991; Xie et al., 1997a; Xie et al., 1997b; FIG. 2). Certain of theboPAGs are useful in detection of early pregnancy in cattle. Thesemolecules are homologous to, but different from, boPAG1 (Xie et al.,1991; FIG. 1; FIG. 3). The inventors now estimate that there are atleast 100 PAG-related genes in cattle, and the inventors have alreadycloned and wholly or partially sequenced at least 20 distinct cDNA(including 10 complete cDNA from early pregnancy). Apparently, PAGsconstitute a polymorphic group (Xie et al., 1994; Xie et al., 1995; Xieet al., 1997a; Xie et al., 1997b), whose members either show variabledegrees of immunocrossreactivity or do not cross-react at all with theantisera that have been developed. Some of the cloned PAGs are onlyexpressed in binucleate cells of the placenta (see Example 3). Thesecells are known to have an endocrine function (Wooding, 1992). Theyproduce placental lactogen and steroids, for example. However, thefunctions of the PAG family members are unknown, although they enter thematernal circulation.

One important aspect of the present invention is that PAGs are notexpressed uniformly throughout pregnancy (see Example 4). Some are foundearly in pregnancy, while are others are expressed in later stages. Forexample, PAGs that are expressed most strongly in the invasivebinucleate cells at implantation are not dominant in late pregnancy.Conversely, boPAG1 (PSP-B) (Xie et al., 1991; Butler et al., 1982;Sasser et al., 1986) primarily is a product of binucleate cells of thelate placenta, and antiserum raised against it fails to recognize thedominant PAG produced by binucleate cells in early pregnancy. Therefore,the test developed by the other groups and based on boPAG1/PSP-B/PSP60(Butler et al., 1982; Sasser et al., 1986; Zoli et al., 1992a; Mialon etal., 1993; Kiracofe et al., 1994 ) is only marginally useful early inpregnancy because the antigen is produced in extremely small amounts, ifat all, at that time. The expression pattern of boPAG1 also helpsexplain the concentration profile of the antigen measured in serum. Atterm, levels can exceed 5 μg/ml, while at day 40, when the developmentof the placenta in terms of size is almost complete, concentrations arearound 10 ng/ml, i.e., 500-fold lower.

Certain of the novel boPAGs disclosed in this invention (boPAG4, 5, 6,7, and 9), having the sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, and SEQ ID NO:32 are present at day 25 ofpregnancy. These PAGs are expressed in invasive binucleate cells whichrelease their secretory granules into maternal uterine capillary bed(see Example 3). Of these five, boPAG4 appears to cross react with thelate pregnancy PAG, boPAG1, which has been the basis of the earlierpregnancy test (see Example 1). By virtue of their early expression,these PAGs can be detected by conventional immunological techniques inphysiological fluids of heifers or cows (especially in serum, urine, andmilk) to detect the presence of a fetus or fetuses in the uterus priorto day 30 of pregnancy. Thus, the presence of these antigens provide adiagnostic test of early pregnancy in cattle.

Similar observations on the diversity of PAGs, the localization ofdifferent PAGs to either mononucleated and binucleated cells, and thelikely varied timing of PAG expression have been noted in sheep (Xie etal., 1991; Xie et al., 1997a; Xie et al., 1997b). Because of the largenumber of genes noted in other species (FIG. 6) these observations arelikely also to hold for other Artiodactyla, as well.

C. Structural, Functional and Evolutionary Aspects of PAGs

PAGs are members of the aspartic proteinase gene family (Xie et al.,1991; Xie et al., 1994; Xie et al., 1995), although the inventors do notbelieve they are necessarily active as proteolytic enzymes. cDNAs forthese antigens (called pregnancy-associated glycoproteins or PAG) havebeen cloned from early placenta and expressed in a variety of systems inorder to produce recombinant products.

The active aspartic proteinases, which include the various pepsins,chymosins, cathepsin E and D and renin, are clustered in the centralbranches of the tree. Included among them is eqPAG1, which is pairedwith rabbit pepsinogen F. EqPAG1 is an active proteinase afterpropeptide excision (Green et al., 1998) and may therefore be the horsehomolog of pepsin F. Unfortunately little is known about pepsinogen F;it has been cloned from the stomach of a neonatal rabbit (Kageyama etal., 1990), but its overall expression pattern in the fetus has not beenstudied, nor has pepsinogen F been described in any other species.

BoPAG1 and 2 occupy an intermediate position between the enzymaticallyfunctional aspartic proteinases and the PAGs from cattle and sheep. Ofthe latter, boPAG8, boPAG10 and ovPAG5 are the three most distant andpossibly most ancient gene products so far identified. Most closelyrelated to them are ovPAG2 and boPAG2, 11 and 12. As determined by insitu hybridization analysis, their genes are expressed in both themononucleated as well as the larger invasive binucleated cells of theouter trophectoderm layer of the placenta. The remaining PAG genes,ovPAG1, 3, 4, 6, 7, 8 and 9 and boPAG1, 3, 4, 5, 6, 7 and 9, which havediverged less than the grouping above, have strictly binucleatecell-specific expression. Because binucleate cells are a typical featureof the trophectoderm of the synepitheliochorial placentas of the pecoranruminants (suborder: Ruminantia) (Wooding, 1992), it is tempting tospeculate that the PAG1related genes diverged relatively recently.

If the entire PAG gene family arose by a series of relatively recentduplications during the diversification of the even toed ungulates(Artiodactyla), the expected lengths of the branches leading to theindividual PAG might be expected to be relatively short. Instead manyare long, far exceeding the distance between human, rabbit and ratcathepsin E (FIG. 8) whose divergence encompasses more than 100 millionyears of evolutionary time. There seem to be two alternativeexplanations. One is that the recent origin theory is wrong and thatduplication of PAGs occurred early in the diversification of mammals.The second is that the genes duplicated late but accumulated mutationsat a high rate. Early diversification seems unlikely in view of the factthat large numbers of aspartic proteinase gene family members have notbeen described in either rodents or man despite considerable efforts toclone them (Birch and Loh, 1991). The inventors' data for the horse(Perissodactyla) and cat (Carnivora) indicate only a limited number (andpossibly only a single) expressed PAG gene in each species. Therefore,the inventors favor a late and rapid diversification of the PAG withinthe Artiodactyla. In this regard, the relatedness of ovPAG2 and boPAG11(94% at the amino acid level) suggests they are functional homologs.These genes are the most closely related of all the PAGs shown in FIG.8, despite a species separation of around 18 million years (Miyamoto etal., 1993).

An analysis (Nei, 1987; Li, 1993) of the nucleotide substitutions withinthe protein-coding regions of the PAG genes reveals that the ratio ofsynonymous (silent) mutations per synonymous site (Ks) to nonsynonymous(replacement) mutations per nonsynonymous site (Ka) in pairwisecomparisons among all PAGs averages 1.18±0.27 (mean±S.D.). A closerexamination indicates that within highly conserved regions the Ks to Karatio is high, while it is low in the hypervariable loop-encodingregions. For example, the Ks to Ka ratio averages 3.07±1.08 for thehighly conserved 29 codons encoding the buried carboxyl end of themolecules. By contrast, the value for the preceding 21 codons, which arehypervariable and encode the two loops (291-296 and 281-287) shown inFIG. 5B, is 0.53±0.18. Thus, mutations that alter amino acids haveaccumulated faster than silent mutations.

Mutations that lead to amino acid changes are much more likely to bedeleterious and therefore to be eliminated than synonymous changes. Forthis reason Ks/Ka ratios are generally greater than 2.0 (Ohta, 1992).The PAGs appear exceptional in this respect, with the data suggestingthat their high variability has occurred as the result of positiveselection. Other related aspartic proteinases, such as ovine and bovinechymosins, enzymes whose coding regions are 95% identical in sequence(Moir et al., 1982; Pungercar et al., 1990) despite 18 million years ofseparation (Miyamoto et al., 1993), exhibit a Ks to Ka ratio of 2.47, avalue more than twice as high as the average PAG pair. The only PAG pairthat exhibits a comparable value to the chymosins is ovPAG2 and boPAG1 I(ratio 2.92) proteins whose relatedness has been commented upon earlier(FIG. 8) and which may be functional homologs. Equine PAG and rabbitpepsinogen F, both active enzymes, provide a value of 2.61. Conceivablythese genes have also acquired a function that is less able to toleratechanges in the surface loop regions than PAGs in general.

In a more general context, the evolution of multigene families has beenthe subject of several recent reviews (Ohta, 1995; Hughes, 1994;Fryxell, 1996). All agree that most duplicated genes are likely eitherto be quickly lost or accumulated as pseudogenes, as a result of“purifying” Darwinian selection, unless they acquire a novel function.By this argument it must be assumed that individual PAGs are not onlyfunctional molecules, but that each has a subtly different role. Hughes(1994) has argued that weak bifunctionality must be acquired prior togene duplication and that, once duplicated, genes become separated by aburst of amino acid replacements that allows a specific function tobecome fixed and enhanced. These mutations are likely to be acquired bya combination of nonsynonymous point mutations, and by gene conversionevents which can probably occur readily between closely linked,structurally similar genes (Ohta, 1995). Genetic drift and naturalselection will ensure the retention of those mutations that are notdeleterious. At present it is not possible to estimate what kinds ofmutational changes contributed most to PAG diversity.

Fryxell (1996) has argued that the retention of a duplicated gene willin general, require the presence of a preexisting or similarly evolvingfamily of complementary molecules with which the products of theduplicated genes can interact. Among the best known rapidly evolvinggene families are immunoglobulins, T cell receptors and MHC antigens,the cytochrome p450 system and the odorant receptors. In each of thesecases, diversification is linked to a more exacting capacity to bindparticular ligands. For the PAGs, it is tempting to speculate that theirfunction relates to their peptide-binding capabilities, although afunction involving some structural feature other than the cleft, such asthe propeptide or carbohydrate, cannot be ruled out. Even though theregions around the two catalytic aspartyl residues are generallyconserved in all aspartic proteinases (Davis, 1990; Takahashi et al.,1995), substitutions elsewhere can markedly influence what peptides gainaccess to the catalytic center, clearly evident when the exceedinglynarrow substrate specificity of renin is compared with that of pepsin A.The reorganization of the combining site of an antibody against anitrophenyl phosphate hapten as it evolved from its gernline precursorled to a 30,000-fold greater affinity for ligand and involved only ahandful of amino acids, many of which were in a surface location andnone of which made direct contact with the ligand (Wedemayer et al.,1997). Small additive changes in the packing of loops provided acombining site able to lock in the hapten with much greater efficiency.Similar events could presumably modify the peptide-binding cleft of PAGsand provide molecules with a considerable range of specificities.

D. Variants of PAGS

It is contemplated that, for various uses, variants of PAGs can beutilized according to the present invention. These changes may improvestability or function, for example, antigenicity or immunoreactivity. Itmay be desirable to create substitutional, insertional or deletionvariants or fusion proteins from the identified PAGs. Deletion variantslack one or more residues of the native protein. Insertional mutantstypically involve the addition of material at a non-terninal point inthe polypeptide. This may include the insertion of an immunoreactiveepitope or simply a single residue. Terminal additions, are fusionproteins. Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide, suchas stability against proteolytic cleavage, without the loss of otherfunctions or properties. Substitutions of this kind may be termed“conservative,” that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological ftnctional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimetic is expected topermit molecular interactions similar to the natural molecule. Theseprinciples may be used, in conjunction with the principles outlineabove, to engineer second generation molecules having many of thenatural properties of PAGs, but with altered and even improvedcharacteristics.

E. Purification of the Proteins

It will be desirable to purify the various PAGs identified by theinventors or variants thereof. Protein purification techniques are wellknown to those of skill in the art. These techniques involve, at onelevel, the crude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number” (i.e., 2-fold, 5-fold,10-fold, 50-fold, 100-fold, 1000-fold, etc.). The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat or acid pH denaturation of contaminating proteins, followed bycentrifugation; chromatography steps such as ion exchange, gelfiltration, reverse phase, hydroxylapatite and affinity chromatography;isoelectric focusing; gel electrophoresis; and combinations of such andother techniques. As is generally known in the art, it is believed thatthe order of conducting the various purification steps may be changed,or that certain steps may be omitted, and still result in a suitablemethod for the preparation of a substantially purified protein orpeptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE and according tohow extensively it is glycosylated (Capaldi et al., 1977). It willtherefore be appreciated that under differing electrophoresisconditions, the apparent molecular weights of purified or partiallypurified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofmin, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related tomolecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

F. Synthetic Peptides

The present invention also describes portions of PAG-related peptidesfor use in various embodiments of the present invention. Because oftheir relatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Short peptide sequences, or libraries of overlappingpeptides, usually from about 6 up to about 35 to 50 amino acids, whichcorrespond to the selected regions described herein, can be readilysynthesized and then screened in screening assays designed to identifyreactive peptides. Alternatively, recombinant DNA technology may beemployed wherein a nucleotide sequence which encodes a peptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

G. Antigen Compositions

The present invention provides for the use of PAGs or peptides asantigens for the generation of polyclonal antisera and monoclonalantibodies for use in the detection of pregnancy. It is envisioned thatsome variant of a PAG, or portions thereof, will be coupled, bonded,bound, conjugated or chemically-linked to one or more agents vialinkers, polylinkers or derivatized amino acids. This may be performedsuch that a bispecific or multivalent composition or vaccine isproduced. It is further envisioned that the methods used in thepreparation of these compositions will be familiar to those of skill inthe art and should be suitable for administration to animals, i.e.,pharmaceutically acceptable. Preferred agents are the carriers such askeyhole limpet hemocyannin (KLH) or glutathione-S-transferase.

In order to formulate PAGs for immunization, one will generally desireto employ appropriate salts and buffers to render the polypeptidesstable. Aqueous compositions of the present invention comprise aneffective amount of the PAG antigen to the host animal, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions may be referred to as inocula. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknow in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The PAGs also may be administered parenterally or intraperitoneally.Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It should be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases; it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the PAGs inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCi solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, preparations should meet applicable sterility, pyrogenicity,general safety and purity standards.

III. Nucleic Acids

A. PAG-Encoding Sequences

The present invention provides, in another embodiment, genes encodingthe various PAG polypeptides. Specifically, those encoding PAG2, PAG4,PAG5, PAG6, PAG7, and PAG9 are envisioned. Those nucleic acid sequencesencoding the proteins having the sequences of SEQ ID NO:25; SEQ IDNO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; and SEQ ID NO:32 areencompassed by the present invention, as are those polynucleotidesdisclosed in SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ IDNO:7; and SEQ ID NO:9. The present invention is not limited in scope tothese genes, however, as one of ordinary skill in the art could, usingthese nucleic acids, readily identify related PAGs in various otherspecies.

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a given “PAG gene” may contain a variety of different bases andyet still produce a corresponding polypeptide that is functionally(i.e., antigenically, immunologically), and in some cases structurally,indistinguishable from the genes disclosed herein.

Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the detectability of PAGs.

Nucleic acids according to the present invention may encode an entirePAG gene, a domain of a PAG that contains a relevant epitope, or anyother fragment of the PAG sequences set forth herein. The nucleic acidmay be derived from genomic DNA, i.e., cloned directly from the genomeof a particular organism. In preferred embodiments, however, the nucleicacid would comprise complementary DNA (cDNA). At a minimum, these andother nucleic acids of the present invention may be used as molecularweight standards in, for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred. It also is contemplated that a given PAGfrom a given species may be represented by natural variants that haveslightly different nucleic acid sequences but, nonetheless, encode thesame protein (see Table 1).

As used in this application, the term “a nucleic acid encoding a PAG”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In preferred embodiments, the invention concernsa nucleic acid sequence essentially as set forth in for example, SEQ IDNO:25; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; or SEQ IDNO:32. The term “as set forth in, for example, SEQ ID NO:25; SEQ IDNO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; or SEQ ID NO:32” meansthat the nucleic acid sequence substantially corresponds to a portion ofSEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; orSEQ ID NO:32 respectively. The term “functionally equivalent codon” isused herein to refer to codons that encode the same amino acid, such asthe six codons for arginine or serine (Table 1), and also refers tocodons that encode biologically equivalent amino acids, as discussed inthe following pages.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of FIG. 1 will be sequences that are “as set forth in FIG.1.” Sequences that are essentially the same as those set forth in FIG. 1may also be functionally defined as sequences that are capable ofhybridizing to a nucleic acid segment containing the complement of FIG.1 under standard conditions.

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin FIG. 1. Nucleic acid sequences that are “complementary” are thosethat are capable of base-pairing according to the standard Watson-Crickcomplementary rules. As used herein, the term “complementary sequences”means nucleic acid sequences that are substantially complementary, asmay be assessed by the same nucleotide comparison set forth above, or asdefined as being capable of hybridizing to the nucleic acid segment ofSEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; or SEQID NO:9 under relatively stringent conditions such as those describedherein. Such sequences may encode the entire PAGs encompassed herein orfinctional or non-functional fragments thereof.

B. PAG-Encoding Fragments

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used,although others are contemplated. Longer polynucleotides arecontemplated as well. Such oligonucleotides will find use, for example,as probes in Southern and Northern blots and as primers in amplificationreactions. These reagents are particularly useful in identifyingstructurally related PAGs.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

As stated above, one method of using probes and primers of the presentinvention is in the search for genes related to the PAG encompassed inthe instant invention or, more particularly, homologs of PAG from otherspecies. The existence of a variety of homologies strongly suggests thatother homologs will be discovered in additional species. Normally, thetarget DNA will be a genomic or cDNA library, although screening mayinvolve analysis of RNA molecules. By varying the stringency ofhybridization, and the region of the probe, different degrees ofhomology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

C. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments, expression vectors may be utilized toproduce PAGs which can then be purified and, for example, be used togenerate antisera or monoclonal antibody with which further studies maybe conducted. Expression requires that appropriate signals be providedin the vectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

In preferred embodiments, the nucleic acid encoding a gene product isunder transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene. Typically, the promoter is selected for highlevel expression, such as lac inducible promoter for use in E. coli,alcohol oxidase for yeast, CMV IE for various mammalian systems, or thepolyhedron promoter for Baculovirus. Other elements includepolyadenylation signals, origins of replication, internal ribosome entrysites (IRES) and selectable markers (e.g., neomycin, puromycin,hygromycin, DHFR, GPT, zeocin and histidinol).

Transfer of expression constructs into cells also is contemplated by thepresent invention. These include calcium phosphate precipitation (Grahamand Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986;Potter et al., 1984), direct microinjection (Harland and Weintraub,1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer etal., 1987), gene bombardment using high velocity microprojectiles (Yanget al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wuand Wu, 1988).

In certain embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis, to integrate into host cell genome and express viral genesstably and efficiently have made them attractive candidates for thetransfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolasand Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The firstviruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). Retroviruses are a group ofsingle-stranded RNA viruses characterized by an ability to convert theirRNA to double-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins, making them attractive candidates for transformationof cells. Other viral vectors may be employed as expression constructsin the present invention. Vectors derived from viruses such as vacciniavirus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

In a further embodiment of the invention, the expression construct (andPAGs) may be entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991).

IV. Generating Antibodies Reactive with PAGs

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a PAG molecule of the present invention, or anyportion thereof. An antibody can be a polyclonal or a monoclonalantibody composition, both of which are preferred embodiments of thepresent invention. Means for preparing and characterizing antibodies arewell known in the art (see, e.g., Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a peptide or polypeptide of the presentinvention and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically an animal used for production of anti-antisera is a non-humananimal including rabbits, mice, rats, hamsters, pigs or horses. Becauseof the relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to PAG-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular PAG of different species may be utilized in other usefulapplications.

In general, both polyclonal and monoclonal antibodies against PAG may beused in a variety of embodiments. For example, they may be employed inantibody cloning protocols to obtain cDNAs or genes encoding other PAGpolypeptides. They may also be used in inhibition studies to analyze theeffects of PAG related peptides in cells or animals. Anti-PAG antibodieswill also be useful in immunolocalization studies to analyze thedistribution of PAG polypeptides during various cellular events, forexample, to determine the cellular or tissue-specific distribution ofPAG polypeptides under different points in the cell cycle. Aparticularly useful application of such antibodies is in purifyingnative or recombinant PAG, for example, using an antibody affinitycolumn. The operation of all such immunological techniques will be knownto those of skill in the art in light of the present disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified PAG. The immunizing composition is administered in amanner effective to stimulate antibody producing cells. Rodents such asmice and rats are preferred animals, however, the use of rabbit, sheepor frog cells is also possible. The use of rats may provide certainadvantages (Goding, 1986), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/l.Ag 4 l Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

V. Assays for PAG Expression in the Detection of Pregnancy

According to the present invention, the present inventors havedetermined that certain PAGs are advantageously expressed in earlystages of pregnancy and, therefore, can be used as markers in thedetection of pregnancy at an early stage. While the present invention isexemplifed in cattle, its extension to other species including sheep(e.g. deer, antelopes, and giraffes), horses (Perissodactyla), and allother ruminant ungulates and even more distantly related species (dogs,cats, humans) is contemplated. In addition, the immunoassays, may bequalitative or quantitative.

In cattle, the boPAGs may be used individually or in combination toprovide a diagnostic evaluation of pregnancy. According to the presentinvention, these boPAGs include BoPAG2, BoPAG4, BoPAGS, BoPAG6, BoPAG7,BoPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16; boPAG17; boPAG18; boPAG19;boPAG20 or boPAG21. Other boPAGs, and PAGs from other species, may proveuseful, alone or in combination, for similiar purposes.

A. Immunologic Detection of Pregnancy

The present invention entails the use of antibodies in the immunologicdetection of PAGs. Various useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Nakamura et al.(1987; incorporated herein by reference). Immunoassays, in their mostsimple and direct sense, are binding assays. Certain preferredimmunoassays are the various types of enzyme linked immunosorbent assays(ELISAs) and radioimmunoassays (RIA). Immunohistochemical detectionusing tissue sections also is particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and Western blotting, dot blotting, FACS analyses, and the like also maybe used in connection with the present invention.

In general, immunobinding methods include obtaining a sample suspectedof containing a protein, peptide or antibody, and contacting the samplewith an antibody or protein or peptide in accordance with the presentinvention, as the case may be, under conditions effective to allow theformation of immunocomplexes. Preferred samples, according to thepresent invention, are fluids, such as milk, urine, blood, serum orsaliva.

Contacting the chosen biological sample with the protein, peptide orantibody under conditions effective and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes) isgenerally a matter of simply adding the composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with PAGs. After this time, thePAG-antibody mixture will be washed to remove any non-specifically boundantibody species, allowing only those antibodies specifically boundwithin the primary immune complexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. Patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

Usually, the primary immune complexes may be detected by means of asecond binding ligand that has binding affinity for the PAG or thePAG-specific first antibody. In these cases, the second binding ligandmay be linked to a detectable label. The second binding ligand is itselfoften an antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under conditions effective and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the PAG or anti-PAG antibody is used to formsecondary immune complexes, as described above. The second bindingligand contains an enzyme capable of processing a substrate to adetectable product and, hence, amplifying signal over time. Afterwashing, the secondary immune complexes are contacted with substrate,permitting detection.

B. ELISA

As a part of the practice of the present invention, the principles of anenzyme-linked immunoassay (ELISA) may be used. ELISA was firstintroduced by Engvall and Perlmann (1971) and has become a powerfulanalytical tool using a variety of protocols (Engvall, 1980; Engvall,1976; Engvall, 1977; Gripenberg et al., 1978; Makler et al., 1981;Sarngadharan et al., 1984). ELISA allows for substances to be passivelyadsorbed to solid supports such as plastic to enable facile handlingunder laboratory conditions. For a comprehensive treatise on ELISA theskilled artisan is referred to “ELISA; Theory and Practise” (Crowther,1995 incorporated herein by reference).

The sensitivity of ELISA methods is dependent on the turnover of theenzyme used and the ease of detection of the product of the enzymereaction. Enhancement of the sensitivity of these assay systems can beachieved by the use of fluorescent and radioactive substrates for theenzymes. Immunoassays encompassed by the present invention include, butare not limited to those described in U.S. Pat. No. 4,367,110 (doublemonoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901 (westernblot). Other assays include immunoprecipitation of labeled ligands andimmunocytochemistry, both in vitro and in vivo.

In a preferred embodiment, the invention comprises a “sandwich” ELISA,where anti-PAG antibodies are immobilized onto a selected surface, suchas a well in a polystyrene microtiter plate or a dipstick. Then, a testcomposition suspected of containing PAGs, e.g., a clinical sample, iscontacted with the surface. After binding and washing to removenon-specifically bound immunocomplexes, the bound antigen may bedetected by a second antibody to the PAG.

In another exemplary ELISA, polypeptides from the sample are immobilizedonto a surface and then contacted with the anti-PAG antibodies. Afterbinding and washing to remove non-specifically bound immune complexes,the bound antibody is detected. Where the initial antibodies are linkedto a detectable label, the primary immune complexes may be detecteddirectly. Alternatively, the immune complexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Another ELISA in which the PAGs are immobilized involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesare added to the wells, allowed to bind to the PAG, and detected bymeans of their label. The amount of PAG in a sample is determined bymixing the sample with the labeled antibodies before or duringincubation with coated wells. The presence of PAG in the sample acts toreduce the amount of antibody available for binding to the well, andthus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. In coating a plate with either antigen or antibody, one willgenerally incubate the wells of the plate with a solution of the antigenor antibody, either overnight or for a specified period of hours. Thewells of the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the control human cancer and/orclinical or biological sample to be tested under conditions effective toallow immune complex (antigen/antibody) formation. Detection of theimmune complex then requires a labeled secondary binding ligand orantibody, or a secondary binding ligand or antibody in conjunction witha labeled tertiary antibody or third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG), evaporated or powdered milk, and phosphate bufferedsaline (PBS)/Tween. These added agents also tend to assist in thereduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 h to 2 h to 4 h, attemperatures preferably on the order of 25° C. to 27° C., or may beovernight at about 4° C. or so.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immunecomplex formation (e.g., incubation for 2 h at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azido-di-(3-ethylbenzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

A variant of ELISA is the enzyme-linked coagulation assay, or ELCA (U.S.Pat. No. 4,668,621), which uses the coagulation cascade combined withthe labeling enzyme RVV-XA as a universal detection system. Theadvantage of this system for the current invention, is that thecoagulation reactions can be performed at physiological pH in thepresence of a wide variety of buffers. It is therefore possible toretain the integrity of complex analytes.

C. Immunohistochemistry

While primarily useful in research contexts, immunohistochemistry may beuseful according to the present invention in identifying new PAGs. Thisinvolves testing of both fresh-frozen and formalin-fixed,paraffin-embedded tissue blocks prepared from study byimmunohistochemistry (IHC). For example, each tissue block consists of50 mg of residual “pulverized” placental tissue. The method of preparingtissue blocks from these particulate specimens has been successfullyused in previous IHC studies of various prognostic factors, e.g., inbreast, and is well known to those of skill in the art (Brown et al.,1990; Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen“pulverized” placental tissue at room temperature in phosphate bufferedsaline (PBS) in small plastic capsules; pelleting the particles bycentrifugation; resuspending them in a viscous embedding medium (OCT);inverting the capsule and pelleting again by centrifugation;snap-freezing in −70° C. isopentane; cutting the plastic capsule andremoving the frozen cylinder of tissue; securing the tissue cylinder ona cryostat microtome chuck; and cutting 25-50 serial sections containingan average of about 500 remarkably intact placental cells.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 h fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and embedding the block in paraffin; and cutting up to 50serial permanent sections.

D. Immunodetection Kits

In further embodiments, the invention provides immunological kits foruse in detecting PAGs in biological samples. Such kits will generallycomprise one or more PAGs or PAG-binding proteins that haveimmunospecificity for various PAGs and for antibodies. Morespecifically, the immunodetection kits will thus comprise, in suitablecontainer means, one or more PAGs, antibodies that bind to PAGs, andantibodies that bind to other antibodies via Fc portions.

In certain embodiments, the PAG or primary anti-PAG antibody may beprovided bound to a solid support, such as a column matrix or well of amicrotitre plate. Alternatively, the support may be provided as aseparate element of the kit.

The immunodetection reagents of the kit may include detectable labelsthat are associated with, or linked to, the given antibody or PAGitself. Detectable labels that are associated with or attached to asecondary binding ligand are also contemplated. Such detectable labelsinclude chemilluminescent or fluorescent molecules (rhodamine,fluorescein, green fluorescent protein, luciferase), radioabels (³H,³⁵S, ³²P, ⁴C, ¹³¹I) or enzymes (alkaline phosphatase, horseradishperoxidase).

The kits may further comprise suitable standards of predeterminedamounts, including both antibodies and PAGs. These may be used toprepare a standard curve for a detection assay.

The kits of the invention, regardless of type, will generally compriseone or more containers into which the biological agents are placed and,preferably, suitable aliquoted. The components of the kits may bepackaged either in aqueous media or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, or even syringe or other containermeans, into which the antibody or antigen may be placed, and preferably,suitably aliquoted. Where a second or third binding ligand or additionalcomponent is provided, the kit will also generally contain a second,third or other additional container into which this ligand or componentmay be placed.

The kits of the present invention will also typically include a meansfor containing the antibody, PAG and any other reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

VI. Methods for Identifying Additional PAGs

By following the basic teachings of the examples, it will be possible toidentify additional PAGs and, further, correlate their expression withearly and late stage pregnancy. This is done by obtaining varioustissues (e.g., placenta) as described in the examples and detecting thepresence of various PAG transcripts therein. One of the best knownnucleic acid amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each ofwhich is incorporated herein by reference in its entirety. These methodsmay be applied directly to the identification of PAGs.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a. DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

Where transcripts are the nucleic acid sample of interest, a reversetranscriptase (RT)-PCR™ amplification procedure may be performed inorder to convert the mRNA transcript to DNA and then amplify it fordetection or cloning. Methods of reverse transcribing RNA into cDNA arewell known and described in Sambrook et al., 1989. Alternative methodsfor reverse transcription utilize thermostable, RNA-dependent DNApolymerases. These methods are described in WO 90/07641 filed Dec. 21,1990. Polymerase chain reaction methodologies are well known in the art.

Using PAG-related sequences as primers for either reverse transcriptionor for amplification, one may selectively amplify PAGs from thesesamples. Alternatively, one may simply create a cDNA library and screenthe library using standard probing formats (e.g., Southern blotting).Identified clones may then be sequenced. Partial clones coding for lessthan a full length transcripts can, in turn, be used to isolate thecomplete sequence from other cDNA or even genomic libraries.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

A. Example 1 Cloning of boPAGs from Placental Tissues Early in Pregnancy

Materials and Methods: Bovine PAG transcripts were cloned from day 19and 25 placentae. RNA from six (Simmunental×Hereford) placentas at day25 of pregnancy was used to construct a cDNA library in λZAPII(Clontech, Palo Alto, Calif.). The library was screened with a mixedprobe of ³²P-labeled bovine, ovine and porcine PAG1 and PAG2, and equinePAG cDNA (Xie et al., 1991, Xie et al., 1994; Xie et al., 1995;Szafranska et al., 1995). The positive clones were isolated and analyzedfor the size of inserts by PCR™ and restriction endonuclease digestion.Sixteen clones of the expected length were partially sequenced. Thesecond screening identified boPAG transcripts that reacted with ananti-boPAG1 antiserum (Zoli et al., 1991; Xie et al., 1991). Duplicatefilter screening was employed to increase the frequency of isolation offull length clones. The first filter was allowed to react with antiserumto identify immunopositive clones (Xie et al., 1991), while the secondfilter was hybridized with a ³²P-labeled probe corresponding to exons1and 2 of boPAG1, ovPAG1 and ovPAG2. The clones positive on both filterswere purified and partially sequenced.

PAG transcripts from a day 19 trophoblast of a Holstein cow were clonedby reverse transcription (RT) and PCR™ procedures. Cellular RNA,extracted from day 19 trophoblast, was first reverse transcribed intocDNA then amplified by PCR™ with a pair of well-conserved primers(boPAGexp3′5′ CCCAAGCTTATGAAGTGGCTTGTGCTCCT3′ (SEQ ID NO:16), andboPAGexp3′ 5′GGGAAGCTTACTTGTCATCGTCGTCCTTGTAGTCGGTACCCACCTGTGCCAGGCCAATCCTGTCATTTC3′ (SEQ ID NO:17). The RT-PCR™ products were clonedinto TA cloning vectors (Invitrogen, CA USA). All the novel boPAG cDNAswere fully sequenced.

Results: Alignment of amino acid sequences of all boPAG available isshown in FIG. 1. BoPAG1, 2 and 3 have been identified previously at Day260 of pregnancy, i.e., close to term (Xie et al., 1991; Xie et al.,1994; Xie et al., 1995) and are, therefore, “late” PAGs. Transcripts forboPAGs 4, 5, 6, 7, 8, 9, 10 and bo PAG11 (SEQ ID NO:4; SEQ ID NO:5; SEQID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ IDNO:11) were all present in the day 25 library (FIG. 3). BoPAG12 (SEQ IDNO:12) was present in the day 19 Holstein placenta (FIG. 3). All theseare, therefore, “early” PAGs and candidates for early pregnancyantigens. Note that PAG2 (SEQ ID NO:2), previously detected in latepregnancy (Xie et al., 1994), is also present at day 19 and 25, but thatboPAG1 (SEQ ID NO:1) is not expressed on any of these days as determinedby a combination of procedures, including immuno screening. This pointis important as the antisera used by others for detecting pregnancy(Sasser et al., 1986; Zoli et al., 1992a; Mialon et al., 1993) appear torecognize boPAG1. Note also that the antiserum against boPAG1 doesrecognize one of the “early” PAGs, namely PAG4. It seems likely,therefore, that these previous investigators were able to detect earlypregnancy in cows because their antiserum fortuitously cross-reacted,albeit weakly, with boPAG4.

A considerable degree of amino acid sequence identity exists among the12 boPAGs listed in FIG. 1. The most related are boPAG1 and boPAG3,sharing a 86% amino acid identity. The least related are boPAG4 andboPAG10 with only 49% identity. Interestingly boPAG1 and boPAG4, whichas noted above cross react with the anti-boPAG1 antiserum, exhibit only76% identity at the amino acid level. Presumably a common epitope existson the two molecules.

The hypervariable regions noted in FIG. 1 coincide with surface loopregions on the modeled structures (Xie et al., 1997b) and are potentialdistinguishing epitopes. In this regard, boPAG1 and boPAG4 share onecommon loop (LSKDEREGS:209-217; PAG1 numbering) (FIG. 1), which mayexplain their immunological cross reactivity. Other loops could bemimicked as synthetic peptides and used to immunize rabbits or mice inorder to raise specific antibodies against particular PAGs.

These data show that boPAG1, the antigen used as the basis for previouspregnancy tests, is a “late” PAG and not ideal as an early pregnancyantigen. The data also show that the “early” PAGs are relativelynumerous and differ considerably from each other and from boPAG1 insequence. These differences are most marked in surface loop regions,which are likely to be the most immunoreactive features of the molecule.

B. Example 2 Structural Relationships Among boPAGS

Materials and Methods: The amino acid sequences of various PAGs andpepsin were assembled into multiple sequence alignments with the Pile UpProgram of the Wisconsin GCG Package, Version 9.0 (Madison, Wis.). Adistance matrix was then created (Program Distances) and a phylogenetictree constructed by a neighbor-joining procedure (Nei, 1987).

Results: The data in FIG. 5 is a phylogenetic tree relating all of thebovine PAGs (FIG. 1) and ovine PAGs (FIG. 2) that have so far beencloned as cDNA. The methods used for cloning these PAG cDNAs isdescribed by Xie et al., 1997b. Also included in FIG. 5 are rabbitpepsinogen F and porcine pepsinogen A, the aspartic proteinasesstructurally most similar to PAGs. Note that the bovine and ovine PAGsfall largely into two structurally related groups. One contains boPAG2,-10, -11, and -12, along with ovPAG2 and ovPAG5. The other is comprisedof boPAG1, 3, 4, 5, 6, 7, and 9. As pointed out below and by Xie et al.,(1997b) the boPAGs in this second group are expressed only in binucleatecells, the invasive component of the trophoblast and the cell typeconsidered to release PAGs into the maternal bloodstream. Note thatamong the PAGs in the second group are the “early” PAGs, boPAG4, 5, 6,7, and 9.

C. Example 3 Certain Early PAGs are Expressed in Trophoblast BinucleateCells and in the Syncytium Formed Between Trophectoderm and UterineEpithelium

Materials and Methods: Riboprobes (cRNA) were prepared by using theRiboprobe Preparation System (Promega, Wis., USA). Briefly, two regionsof the boPAG cDNA, representing poorly conserved sequences, were used asthe probe in situ hybridization (and ribonuclease protection assay: seenext section). The first fragment (536bp) of boPAG2, 4, 8, 9 or 11 cDNA,that was in the region of exons 6, 7, 8 and 9, was amplified by usingPCR™ with a pair of primers (Forward 5′CCTCTTTTGCCTTCTACTTGA3′ (SEQ IDNO: 18, and Reverse 5′GCGCTCGAGTTACACTGCCCGTGCCAGGC3′ (SEQ ID NO:19).However, another region (407bp) was chosen for boPAG1, 5, 6 and 7 cDNA,corresponding to exons 3, 4 and 5. Again it was amplified by a PCR™procedure with two well conserved primers (Forward B:5′TGGGTAACATCACCATTGGAA3′ (SEQ ID NO:20, Reverse B:5′TTTCTGAGCCTGTTTTTGCC5′ (SEQ ID NO:21). The PCR™ products weresubcloned into TA cloning vectors (Invitrogen, CA, USA). The orientationand sequence of the inserts were determined by sequencing.

The subcloned cDNA fragments were then transcribed in vitro into cRNA inthe presence of [³⁵S]-CTP. Non-incorporated [³⁵S]CTP was removed bycentrifugation of the labeling mix through a Sephadex G-50 column. Thecontrol probes, sense cRNA of boPAG, were prepared in essentially theway described above. The probes were used within 3 days. Day 25 or Day100 tissue was sectioned (14μm) at −18 ° C. with an IEC cryostat(International Equipment Co., Needham Heights, Mass.) and mounted ontoprechilled microscope slides.

The sections were then fixed and processed as described by Xu et al.,(1995). Hybridization was performed by application of about 200 μl ofprobe solutions (4×10⁶ cpm) to cover each section and incubated at 55°C. for 12 to 18 h. After hybridization, the slides were dipped in 2×SSCto remove the excess hybridization buffer, treated with RNase A(50 μl/mlin PBS) for 30 min at 37° C. to eliminate probes that were nothybridized. The sections were then washed at 55° C. in 2×SSC for 15 min,in 50% formamide in 2×SSC for 30 min and twice in 0.1×SSC for 15 min.Slides were again dehydrated, air dried, coated with Kodak NTB-2emulsion (Eastman Kodak, Rochester, N.Y.) and exposed for 1 to 4 weeksat 4° C. Finally, the slides were developed, counterstained withhematoxylin and eosin and examined microscopically.

In situ hybridization was performed with [³⁵S]-antisense probes onsections through placentomes (areas of fused cotyledonary, i.e., fetaland caruncular, i.e., maternal, villi). Resulting autoradiographs werestained with hemotoxylin and cosin and photographed. No specifichybridization signals were shown with sense probe. BoPAG9 mRNA wasconcentrated in the more scattered binucleate cells, while that forboPAG11 was found in all the cells of the chorionic epithelium(trophectoderm).

In situ hybridization was performed with [³⁵S]-antisense probes on day25 endometrium-placental sections using darkfield micrographs at 20× and40×. The silver-grains appear to be white dots under darkgroundillumination. The cell layer at the edge of the section gave an intenseboPAG6 signal. Abundant silver stains were localized to the cells at themargin of the section. In contrast, boPAG2 mRNA gave only a weak signalwithin the syncytial region. Few silver grains were visible at the edgeof the section.

Results:

1. Localization of boPAG mRNAs at Day 100 of Pregnancy

The outer layer of the placenta consists of two populations oftrophoblast cells, mono- and binucleate trophoblast cells. To localizethe site of each PAG expression specifically to mono- or binucleatetrophoblast cells, in situ hybridization were performed to detectindividual PAG mRNA. Previous published data have shown that whileboPAG1 is expressed in trophoblast binucleate cells (Xie et al., 1994),boPAG2 is expressed throughout the trophectodern, including the moreabundant mononucleated cells that comprise 80% or more of the epithelium(Xie et al., 1994).

Here, in situ hybridization has been on sections of placentomes employedto determine in what cell type the remaining characterized boPAGs areexpressed. BoPAG9 is expressed largely in the scattered binucleatecells, which are heavily covered with silver grains. By contrast, mRNAfor boPAG11 is found throughout the epithelium covering the cotyledonaryvilli.

There is a correspondence between the PAGs that are expressed inbinucleate cells and their positions in the phylogenetic tree (FIG. 5),and that four of the PAGs known to be expressed early, namely boPAG4, 5,6, 7 and 9, are produced by the invasive binucleate cell, and therefore,likely to enter the maternal bloodstream.

2. Localization at Day 25 of Pregnancy

Bovine placenta on day 25 of pregnancy is not fully developed and thecotyledons are not firmly interdigitated with the caruncularendometrium. Therefore, the thickened placental membrane was processedwith the attached endometrium. By the time it had been through the insitu hybridization procedures, most of the membrane was lost. Only thelayer that fused with the endometrium survived the harsh procedure andremained on the surface of endometrium.

It was very difficult to identify individual cells since most cells (theremaining placental tissue) were fused with the underlining endometrialcells. Nevertheless, these fused multicellular syncytium containedplentiful amount of boPAG6 mRNA. As observed previously, only binucleatetrophoblast can fuse with endometrium. Therefore, the placental cells inthe syncytium are most likely to be binucleate trophoblast cells inorigin. Similarly the sections hybridized to boPAG4, 5, 7 and 9 probes,also had very strong signals at the interface between the remainingplacental membrane and the endometrial epithelium. Hence, they are mostlikely to be expressed by the binucleate trophoblast cells.

In contrast, very little mRNA for boPAG2, 8, 10, 11 was localized to thesyncytical layer. A plausible explanation is that either those boPAG arenot expressed or are expressed at low levels in the fused binucleatetrophoblast cells at day 25 placenta. They are less likely, therefore,to be found in maternal blood than boPAG4, 5, 6, 7 and 9.

D. Example 4 Relative Expression of mRNA for Different boPAG TranscriptsVaries Over Gestation in Cows

Materials and Methods: Riboprobes (cRNA) were prepared by the RiboprobePreparation System (Promega, Wis., USA). Briefly, two regions of theboPAG cDNA, that represent poorly conserved regions of PAGs in generalwere used as probes for RPA as well as for in situ hybridization. Thefirst fragment (536 bp) of boPAG2, 4, 8, 9 and 11 cDNA, in the region ofexons 6, 7, 8 and 9, was amplified by using PCR™ with the same pair ofprimers (SEQ ID NO:18 and SEQ ID NO:19) described in Example 3 for insitu hybridization. Similarly a region (407 bp) of boPAG1, 5, 6, or 7cDNA corresponding to exons 3, 4 and 5, was amplified as described inExample 3 with primers (SEQ ID NO:20 and SEQ ID NO:21).

After subcloning, the cDNA fragments were transcribed in vitro into cRNAin the presence of [³²P-α]CTP. Total cellular RNA was extracted fromplacental tissue at different stages of pregnancy by using guanidiumisothiocyanate and purified over a cesium chloride gradient (Sambrook etal., 1989; Ausubel et al., 1987). Twenty μg of RNA was used for each RPAreaction according to the manufacturer's recommendations (Ambion Inc.,Austin, Tex.). In short, the sample RNA was co-precipitated with³²P-labeled probes 2×10⁶ cpm/sample) and the pellet suspended in 10 μlof hybridization buffer and incubated at 68° C. for 10 min. UnhybridizedcRNA was digested with a mixture of RNase A/TI for 45 min at 37° C. ThecRNA probe and mRNA hybrids were precipitated and separated in 6% longrange sequence gels and visualized by autoradiography.

A fragment of boPAG cDNA was amplified by PCR™ and the productssubsequently subcloned into TA cloning vectors. Those fragments werethen in vitro transcribed into riboprobes in the presence [³²P]CTP. RNAwas extracted from bovine conceptus and placenta on days 25, 45, 88, 250and term of pregnancy. The total tissue RNA (20 μm) was then hybridizedwith cRNA probes of boPAG1, boPAG2, boPAG4, boPAG5, boPAG6, boPAG7,boPAG8, boPAG9, boPAG10 and boPAG11. The protected DNA fragments wereseparated and visualized by autoradiography.

Results: The length of gestation in cattle is about 285 days. Initialimmunoscreening of cDNA libraries previously identified three boPAG(boPAG1, 2 and 3). More recently two additional cDNA (boPAG13 andboPAG14) were cloned from mRNa of term placenta by using hybridizationscreening (SEQ ID NO: 13) and (SEQ ID NO:14) in a day 260 placental cDNAlibrary (Xie et al., 1991; Xie et al., 1995). On day 25 pregnancy, tendistant PAG were identified (Example 1, FIG. 1, FIG. 2 and FIG. 5). OnlyboPAG2 was isolated from both stages of pregnancy. These cloning dataimply that expression of individual boPAG is temporally controlled. Toconfirm the temporal expression of boPAG, ribonuclease protection assayswere carried out to delineate the stages at which individual boPAGgeneswere expressed in the cattle placenta. This procedure was repeated atleast twice for each boPAG riboprobe and for each RNA sample. The majorband represents the protected boPAG mRNA. In addition, there weremultiple small bands in each lane. Those smaller bands almost certainlyprotected sequences highly related to, but distinct from, that of theriboprobe.

In summary boPAG2, was found in RNA at days 19, 25 and 260 and wastherefore expressed through gestation. Similarly boPAG8, 10 and 11 wereexpressed at all stages of pregnancy examined. BoPAG1, which wasoriginally characterized from day 260 placenta and is the basis of thepregnancy test of Sasser et al., (1986), Zoli et al., (1992a) and Mialonet al., (1992; 1993) was expressed at a very low level on day 25 ofpregnancy. By day 45, its expression was elevated markedly. Other boPAGin the same group had varied expression on day 25. However, none of themshowed enhanced expression by day 45 of pregnancy.

E. Example 5 Artiodactyla Species Related to Bos taurus Also HaveMultiple PAG Genes

Materials and Methods: Southern genomic blots of bovine DNA wereperformed with probes corresponding to a segment of the boPAG1encompassing part of intron 6, exon 7, intron 7, exon 8 the proximal endand the proximal end of intron 8 (Xie et al., 1995). The restrictionenzyme EcoRI was chosen that did not cleave the probe. Conditions ofhybridization were such that the PAG1 probe did not bind the PAG2 gene,nor would there be hybridization to genes for other known asparticproteinases.

Results: Multiple PAG genes were detectable in all species of theBovidae family examined. Signals were especially strong in the speciesclosely related to Bos taurus within the subfamilies Bovinae (e.g., Bosfrontalis gaurus, gaur; Bos grunniens, yak: Syncerus caffer, Capebuffalo) and Caprinae (e.g., Ovis aires, domestic sheep; ovis dalli,Dall sheep; Capra falconeri, Markhor goat, Nemorhaedus goral, goral;Budorcas taxicolor, takin). Gazelle and antelope species in otherrelated subfamilies, including the impala, gnu, duiker, and nyala, alsogave strong signals.

In general hybridization, although detectable, was weaker to DNA ofmembers of the Cervidae family, including the whitetail deer and muledeer, than to DNA from Bovidae. Unexpectedly, moose (Alces alces) gave arelatively strong signal. The giraffe (family Giraffidae) provided theweakest signal of the true pecoran ruminants, possibly reflecting itsearly divergence (Kageyama et al., 1990). Hybridization to DNA from theNile hippopotamus was barely detectable with the boPAG1 probe employed.since the hippo (family Hippotamidae; suborder Suiformes) is related tothe domestic pig (Sus Scrofa), a species with multiple PAGs (Szafranskaet al., 1995), this result indicates the considerable divergence of thegenes within the Artiodactyla order over the 55 to 65 million years ofits existence.

These data together show that there are multiple PAG genes withconsiderable structural similarity to boPAG1 in all ruminant ungulatespecies examined. Thus, a pregnancy test developed for domestic cattle(Bos taurus) on the basis of “early” PAG secretion by the placenta mightalso have utility in these other species as well.

F. Example 6 The Placenta of the Domestic Cat (Feli catus) Expresses aPAG Related to boPAGs

Materials and Methods: Day 30 cat placentas from a single litter wereobtained from the University of Missouri Veterinary Taching Hospital.Tissue was cut into small chunks and frozen in liquid N₂. Total RNA wasextracted from frozen tissues and polyA⁺ mRNA purified by using themicro-FastTrack™ kit from Invitrogen, CA. This RNA was reversetranscribed and the resulting cDNA collected ( ). PCR™ was conductedwith the following primers, which represent highly conserved regions ofthe majority of boPAG genes (5′TGGGTAACATCACCATTGGAAC3′ (215-236), (SEQID NO:22, ovPAGe5r 5′CAAACATCACCACACTGCCCTCC3′ (667-645), (SEQ IDNO:23).

PCR™ reactions were run for 35 cycles. Each cycle was 94° C. for1 min.;42° C. for 1 min.; 72° C. for 1 min. The TA cloning kit (Invitrogen, CA)was employed to clone the PCR™ products. Plasmid DNA was isolated byusing a Mini Prep Kit (Promega, Madison, Wis.). The isolated plasmid DNAwere digested with the EcoRI restriction enzyme to check the sizes ofinserts. In order to localize the site of cat PAG expression moreprecisely, in situ hybridization (as described in Example 3, section C)was used to detect cat PAG mRNA in frozen day 30 cat placental tissue.Cat PAG transcripts were detected with an antisense ³⁵S-labeledriboprobe.

Results: The open reading frame of the cat PAG cDNA was 1164bp andencoded a polypeptide of 388 amino acids with a predicted Mr of 43,035Cat PAG (SEQ ID NO:15). The amino acid sequence (SEQ ID NO:38) of catPAG showed between 50 and 60% identity to all known bovine PAGs and59.4% identity to porcine pepsinogen A.

Together these data suggest that the PAG occur outside the Ungulataorder and are also found in non-hoofed species such as the domestic cat.By inference they are likely to be also found in related cat species(Felidae) as well as in the dogs (Canidae). A pregnancy test based on“early” PAG antigens could have utility in these species, particularlyin the domestic dog (Canis familiarus).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

VIII. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A method for detecting pregnancy in a bovine animal comprising: (a)obtaining a sample from said animal; and (b) contacting said sample withan antibody that binds immunologically to at least one pregnancyassociated antigen (PAG), wherein said PAG is present in early pregnancyand undetectable at about two months post-partum, and wherein said PAGis selected from the group consisting of PAG4 (SEQ ID NO:27), PAG6 (SEQID NO:29), PAG7 (SEQ ID NO:30), PAG16 (SEQ ID NO:46); PAG17 (SEQ IDNO:29), PAG7 (SEQ ID NO:30), PAG16 (SEQ ID NO:46); PAG17 (SEQ ID NO:48);PAG20 (SEQ ID NO:54) and PAG21 (SEQ ID NO:56); and (c) detecting saidPAG bound to said antibody; whereby the presence of said PAG in saidsample indicates that said animal is pregnant.
 2. The method of claim 1,wherein said sample is saliva, serum, blood, milk or urine.
 3. Themethod of claim 2, wherein said sample is saliva.
 4. The method of claim2, wherein said sample is serum.
 5. The method of claim 2, wherein saidsample is blood.
 6. The method of claim 2, wherein said sample is milk.7. The method of claim 2, wheren said sample is urine.
 8. The method ofclaim 1, wherein said detection comprises detection with polyclonalantisera.
 9. The method of claim 1, wherein said detection comprisesdetection with a monoclonal antibody preparation.
 10. The method ofclaim 1, wherein said detection comprises ELISA.
 11. The method of claim1, wherein said detection comprises RIA.
 12. The method of claim 1,wherein said detection comprises Western blot.
 13. The method of claim1, further comprising detecting a second PAG in said sample.
 14. Themethod of claim 13, rther comprising detecting a third PAG in saidsample.
 15. The method of claim 10, wherein said ELISA is a sandwichELISA comprising binding of a PAG to a first antibody preparation fixedto a substrate and a second antibody preparation labeled with an enzyme.16. The method of claim 15, wherein said enzyme is alkaline phosphataseor horseradish peroxidase.
 17. The method of claim 15, wherein saidfirst antibody preparation is monoclonal.
 18. The method of claim 1,wherein said PAG is PAG4 (SEQ ID NO:27).
 19. The method of claim 1,wherein said PAG is PAG6 (SEQ ID NO:29).
 20. The method of claim 1,wherein said PAG is PAG7 (SEQ ID NO:30).
 21. The method of claim 1,wherein said PAG is PAG16 (SEQ ID NO:46).
 22. The method of claim 1,wherein said PAG is PAG17 (SEQ ID NO:48).
 23. The method of claim 1,wherein said PAG is PAG20 (SEQ ID NO:54).
 24. The method of claim 1,wherein said PAG is PAG21 (SEQ ID NO:56).